A deep sequencing tool for partitioning clearance rates following antimalarial treatment in polyclonal infections

Background and objectives: Current tools struggle to detect drug-resistant malaria parasites when infections contain multiple parasite clones, which is the norm in high transmission settings in Africa. Our aim was to develop and apply an approach for detecting resistance that overcomes the challenges of polyclonal infections without requiring a genetic marker for resistance.Methodology: Clinical samples from patients treated with artemisinin combination therapy were collected from Tanzania and Cambodia. By deeply sequencing a hypervariable locus, we quantified the relative abundance of parasite subpopulations (defined by haplotypes of that locus) within infections and revealed evolutionary dynamics during treatment. Slow clearance is a phenotypic, clinical marker of artemisinin resistance; we analyzed variation in clearance rates within infections by fitting parasite clearance curves to subpopulation data.Results: In Tanzania, we found substantial variation in clearance rates within individual patients. Some parasite subpopulations cleared as slowly as resistant parasites observed in Cambodia. We evaluated possible explanations for these data, including resistance to drugs. Assuming slow clearance was a stable phenotype of subpopulations, simulations predicted that modest increases in their frequency could substantially increase time to cure.Conclusions and implications: By characterizing parasite subpopulations within patients, our method can detect rare, slow clearing parasites in vivo whose phenotypic effects would otherwise be masked. Since our approach can be applied to polyclonal infections even when the genetics underlying resistance are unknown, it could aid in monitoring the emergence of artemisinin resistance. Our application to Tanzanian samples uncovers rare subpopulations with worrying phenotypes for closer examination.     

Main partitioning criteria for the characterization of the health status in the freshwater mussel Anodonta cygnea from spontaneously polluted area in Western Ukraine

The aim of this study was to appreciate the consequences of spontaneous human activity for freshwater mollusks in the generally ecologically sustainable area in Western Ukraine. For this, bivalve mollusk, Anodonta cygnea, at three sites, with mixed agricultural and municipal activities (A), close to a municipal water inlet (F) and the cooling pond of a nuclear power plant (N), were studied in spring, summer, and autumn. The set of parameters included the characteristics of oxidative stress (activity of catalase (CAT), levels of protein carbonyls (PC)), levels of reduced and oxidized glutathione (GSH, GSSG, respectively), activities of lactate dehydrogenase (LD), cholinesterase (ChE), ethoxyresorufin‐O‐deethylase (EROD), glutathione S‐transferase (GST) in the digestive gland, and concentrations of vitellogenin‐like proteins (Vtg‐LP) in gonads and also morphological indices. Although the discriminant functional analysis confirmed the general seasonal regularities for studied groups, it allowed to discriminate between sites (P < 0.05). At site A, oxidative stress; high levels of LD, EROD, and GST; and low levels of ChE and condition factor were reflected. This demonstrated the sensitivity of mussels to constant effect of mixed pollution. At site N, oxidative injury was shown that might be explained by the constantly high temperature. At site F, abrupt elevations of Vtg‐LP and EROD levels in autumn were probably related to an emergency situation on the nearby dump. So, both chronic and temporal environmental effects were reflected by a set of markers in mollusk. The classification and regression tree (CART) algorithm selected GSH and PC in the digestive gland and Vtg‐LP as partitioning criteria for the characterization of mussel health status. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.     

Analytical approaches for monitoring exposure to organophosphorus and carbamate agents through analysis of protein adducts

Appropriate treatment of a poisoned patient requires knowing the identity of the poison. This review summarizes the methods for identifying poisoning by organophosphorus and carbamate poisons. Mass spectrometry methods identify the poison from the adducts they form with proteins in blood. The most sensitive method uses potassium fluoride to release the organophosphorus agent from its covalent binding to serine 198 of human butyrylcholinesterase. The released poison is identified by gas chromatography-mass spectrometry. The drawback of this method is that it does not detect exposure to agents such as soman, because butyrylcholinesterase adducts with these agents age to a non-reactivatable form. A method that detects both aged and non-aged organophosphylated adducts as well as carbamate adducts is one that digests butyrylcholinesterase with a protease and analyzes the modified peptide by mass spectrometry. This method does not distinguish between poisons that have the same mass after reaction with butyrylcholinesterase--for example, between exposure to chlorpyrifos oxon and paraoxon. Albumin forms a stable, covalent bond with organophosphates on tyrosine 411. The rate of reaction with albumin is much slower than with butyrylcholinesterase, but albumin adducts persist for a longer time in the circulation; they do not age; and they do not release the organophosphate when a patient is treated with an oxime.     

Highly potent analgesic activity of monoterpene-derived (2S,4aR,8R,8aR)-2-aryl-4,7-dimethyl-3,4,4a,5,8,8a-hexahydro-2H-chromene-4,8-diols

New chiral heterocyclic compounds with a hexahydro-2H-chromene framework were synthesized by reactions of (1R,2R,6S)-3-methyl-6-(prop-1-en-2-yl)cyclohex-3-ene-1,2-diol with aromatic aldehydes in the presence of montmorillonite clay. The analgesic activity of the compounds was studied in vivo. The majority of these compounds showed significant analgesic activity in the acetic acid-induced writhing test; the compounds containing one hydroxy and one methoxy substituents also showed analgesic activity in the hot-plate test. (2S,4aR,8R,8aR)-2-(3-Hydroxy-4-methoxyphenyl)-4,7-dimethyl-3,4,4a,5,8,8a-hexahydro-2H-chromene-4,8-diol was as effective as the diclofenac sodium reference taken in the same dose in both tests. It has low acute toxicity and is very promising for further development.     

Protection from the toxicity of diisopropylfluorophosphate by adeno-associated virus expressing acetylcholinesterase

Organophosphorus esters (OP) are highly toxic chemicals used as pesticides and nerve agents. Their acute toxicity is attributed to inhibition of acetylcholinesterase (AChE, EC 3.1.1.7) in nerve synapses. Our goal was to find a new therapeutic for protection against OP toxicity. We used a gene therapy vector, adeno-associated virus serotype 2 (AAV-2), to deliver murine AChE to AChE-/- mice that have no endogenous AChE activity. The vector encoded the most abundant form of AChE: exons 2, 3, 4, and 6. Two-day old animals, with an immature immune system, were injected. AChE delivered intravenously was expressed up to 5 months in plasma, liver, heart, and lung, at 5-15% of the level in untreated wild-type mice. A few mice formed antibodies, but antibodies did not block AChE activity. The plasma AChE was a mixture of dimers and tetramers. AChE delivered intramuscularly had 40-fold higher activity levels than in wild-type muscle. None of the AChE was collagen-tailed. No retrograde transport through the motor neurons to the central nervous system was detected. AChE delivered intrastriatally assembled into tetramers. In brain, the AAV-2 vector transduced neurons, but not astrocytes and microglia. Vector-treated AChE-/- mice lived longer than saline-treated controls. AChE-/- mice were protected from diisopropylfluorophosphate-induced respiratory failure when the vector was delivered intravenously, but not intrastriatally. Since vector-treated animals had no AChE activity in diaphragm muscle, protection from respiratory failure came from AChE in other tissues. We conclude that AChE scavenged OP and in this way protected the activity of butyrylcholinesterase (BChE, EC 3.1.1.8) in motor endplates.     

The Role of Community Health Workers Within the Continuum of Services for HIV,Viral Hepatitis,and Other STIs Amongst Men Who Have Sex with Men in Europe

Journal of Community Health - Little is known about Community Health Workers (CHWs) who work in non-clinical settings to provide sexual health support around HIV, viral hepatitis, and other...     

Ranking Itraconazole Formulations Based on the Flux through Artificial Lipophilic Membrane

Purpose

The goal of the study was to evaluate a miniaturized dissolution-permeation apparatus (μFLUX? apparatus) for its ability to benchmark several itraconazole (ITZ) formulations for which in vivo PK data was available in the literature.

Method

Untreated and micronized powders of ITZ and various enabling formulations of ITZ (commercial Sporanox® solid dispersion, a Soluplus®-based solid dispersion and a nanosuspension) were introduced to the donor compartment of μFLUX? apparatus. Donor and acceptor chambers were divided from each other by a lipophilic membrane. In addition to the flux evaluations, changes in solid state as a function of time were investigated to gain further insight into the flux changes observed over time for the solid dispersion formulations.

Results

Initial flux values from Sporanox®, the nanosuspension and the micronized ITZ showed ratios of 52/4/1 with a decreasing flux from nanosuspension and both solid dispersions after 2.5–3 h. Although the initial flux from the Soluplus® formulation was 2.2 times lower than the one observed for Sporanox®, the decrease in flux observed was milder and became ~ 2 times higher than Sporanox® after approximately 2.5 h. The total amounts of ITZ in the receiver compartment after 240 min showed the same rank order as the rodent AUCs of these formulations reported in literature.

Conclusions

It was demonstrated that in vitro flux measurements using lipophilic artificial membranes could correctly reproduce the rank order of PK results for ITZ formulations. The drop in flux over time for solid dispersions could be backed by experimental indications of crystallization.

     

Methamidophos,dichlorvos, O‐methoate and diazinon pesticides used in Turkey make a covalent bond with butyrylcholinesterase detected by mass spectrometry

Organophosphorus pesticides used most commonly in Turkey include methamidophos, dichlorvos, O‐methoate and diazinon. These toxic chemicals or their metabolites make a covalent bond with the active site serine of butyrylcholinesterase. Our goal was to identify the adducts that result from the reaction of human butyrylcholinesterase with these pesticides. Highly purified human butyrylcholinesterase was treated with a 20‐fold molar excess of pesticide. The protein was denatured by boiling and digested with trypsin. MS and MSMS spectra of HPLC‐purified peptides were acquired on a MALDI‐TOF‐TOF 4800 mass spectrometer. It was found that methamidophos added a mass of +93, consistent with addition of methoxy aminophosphate. A minor amount of adduct with an added mass of +109 was also found. Dichlorvos and O‐methoate both made dimethoxyphosphate (+108) and monomethoxyphosphate adducts (+94). Diazinon gave a novel adduct with an added mass of +152 consistent with diethoxythiophosphate. Inhibition of enzyme activity in the presence of diazinon developed slowly (15 h), concomitant with isomerization of diazinon via a thiono‐thiolo rearrangement. The isomer of diazinon yielded diethoxyphosphate and monoethoxyphosphate adducts with added masses of +136 and +108. MSMS spectra confirmed that each of the pesticides studied made a covalent bond with serine 198 of butyrylcholinesterase. These results can be used to identify the class of pesticides to which a patient was exposed.     

Herbal treatment following post-seizure induction in rat by lithium pilocarpine: Scutellaria lateriflora (Skullcap), Gelsemium sempervirens (Gelsemium) and Datura stramonium (Jimson Weed) may prevent development of spontaneous seizures

About 1 week after the induction of status epilepticus in male rats by a single systemic injection of lithium (3 mEq/kg) and pilocarpine (30 g/kg), rats were continuously administered one of three herbal treatments through the water supply for 30 days. A fourth group received colloidal minerals and diluted food grade hydrogen peroxide in tap water, while a fifth group of rats received only tap water (control). Herbal treatments were selected for their historical antiseizure activities and sedative actions on the nervous system. The numbers of spontaneous seizures per day during a 15 min observation interval were recorded for each rat during the treatment period and during an additional 30 days when only tap water was given. Rats that received a weak solution of the three herbal fluid extracts of Scutellaria lateri flora (Skullcap), Gelsemium sempervirens (Gelsemium) and Datura stramonium (Jimson Weed) displayed no seizures during treatment while all the other groups were not seizure-free. However, when this treatment was removed, the rats in this group displayed numbers of spontaneous seizures comparable to the controls. Although there is no proof that herbal remedies can control limbic or temporal lobe epilepsy, the results of this experiment strongly suggest that the appropriate combination of herbal compounds may be helpful as adjunctive interventions.     

The angiopoietin/Tie-2 system regulates pericyte survival and recruitment in diabetic retinopathy

PURPOSE: The angiopoietin (Ang) system plays an important role in vascular stabilization and pathologic neovascularization. The hypothesis for the study was that, in addition to modulating endothelial cell behavior, the angiopoietin/Tie-2 system also regulates the pericyte apoptosis and/or the vessel maturation associated with diabetic retinopathy. METHODS: Tie-2 expression in cultured retinal pericytes was analyzed by using ELISA, Western Blot analysis, and flow cytometry. CD13 (aminopeptidase N) expression in pericytes was determined by Western blot analysis and Ang effects verified with Tie-2 antisense treatment. Cell proliferation was monitored by crystal violet uptake, and pericyte migration was assessed in a scrape wound. Annexin V-FITC flow cytometry was used to quantify pericyte apoptosis. RESULTS: Pericytes expressed a functionally active Tie-2 receptor upregulated by both Ang-1 and -2 (P < 0.05). In pericytes undergoing apoptosis induced by either TNF-alpha or high glucose Ang-1 increased survival (P < 0.05 for TNF-alpha; P < 0.01 for high glucose), whereas Ang-2 increased apoptosis. Ang-1 enhanced CD13 expression in a dose-dependent manner (P < 0.05) and stimulated pericyte migration in a synthetic matrix wound-healing assay that was associated with a change in F-actin organization. Addition of Tie-2 antisense confirmed that angiopoietins act through Tie-2. CONCLUSIONS: These findings demonstrate that Tie-2 is functional in pericytes and may play an important role in the progression of diabetic retinopathy, by regulating pericyte loss and influencing the activation state and recruitment of pericytes.